A rapidly emerging area of interest in high-pressure environments is that of pupillometry, where handheld quantitative infrared pupillometers (HQIPs) are able to track psycho-physiological fatigue in a fast, objective, valid, reliable, and non-invasive manner. However, the application of HQIPs in the context of athlete monitoring is yet to be determined. Therefore, the main aim of this pilot study was to examine the potential usefulness of a HQIP to monitor game-induced fatigue inside a professional female basketball setting by determining its (1) test-retest repeatability, (2) relationship with other biomarkers of game-induced fatigue, and (3) time-course from rested to fatigued states. A non-ophthalmologic practitioner performed a standardized Pupil Light Reflex (PLR) test using a medically graded HQIP among 9 professional female basketball players (2020–2021 Euro Cup) at baseline, 24-h pre-game (GD-1), 24-h post-game (GD+1) and 48-h post-game (GD+2). This was repeated over four subsequent games, equalling a total of 351 observations per eye. Two out of seven pupillometrics displayed good ICCs (0.95–0.99) (MinD and MaxD). Strong significant relationships were found between MaxD, MinD, and all registered biomarkers of game-induced fatigue (r = 0.69–0.82, p < 0.05), as well as between CV, MCV, and cognitive, lower-extremity muscle, and physiological fatigue markers (r = 0.74–0.76, p < 0.05). Three pupillometrics were able to detect a significant difference between rested and fatigued states. In particular, PC (right) (F = 5.173,
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= 0.115 p = 0.028) and MCV (right) (F = 3.976,
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= 0.090 p = 0.049) significantly decreased from baseline to GD+2, and LAT (left) (F = 4.023,
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= 0.109 p = 0.009) significantly increased from GD-1 to GD+2. HQIPs have opened a new window of opportunity for monitoring game-induced fatigue in professional female basketball players. However, future research initiatives across larger and heterogenous samples, and longer investigation periods, are required to expand upon these preliminary findings.